Human embryonic stem cells (hESC's) (markers for hESCs include SSEA3, SSEA4, TRA-1-60, TRA-1-81 antigens, Nanog, Oct4) are a pluripotent population of cells that can be differentiated into cells derived from all three embryonic germ layers and extraembryonic lineages. FIG. 33. This property of hESC's has important implications in cell therapy (e.g. diabetes, heart disease, neurodegenerative diseases), drug discovery and developmental modeling.
Other pluripotent cell types have been identified in mouse. Primitive ectoderm like (EPL; Rathjen et al., 1999, J. Cell Sci) cells were shown to form from mESC's with the ability to dedifferentiate into mESC's. Recently, a new mouse cell, post-implantation epiblast stem cells (EpiSC; Tesar et al., Nature 448: 196-202; 2007) was identified that shares characteristics of hESC's (Nanog+ Sox2+ Oct4+). All of these pluripotent cell types from mouse can generate the three embryonic germ layer in vitro or in a teratoma assay.
Epiblast stem cells (EpiScs) and induced pluripotent stem cells (iPS) fit into the broad pluripotent cell category and in concept, the technology described in the application could apply to these and other pluripotent cell types (ie, primate pluripotent cells). EpiSc epiblast stem cells are isolated from early post-implantation stage embryos and express Oct4 and are pluripotent (Tesar et al, Nature, Vol 448, p. 196 12 Jul. 2007). Induced pluripotent stem cells (iPS cells) are made by dedifferentiating adult skin fibroblasts or, other adult somatic cells, back to a pluripotent state by retroviral transduction of four genes (c-myc, Klf4, Sox2, Oct4) (Takahashi and Yamanaka, Cell 126, 663-676, Aug. 25, 2006).
The advantage of developing other non-ESC, self renewing, pluripotent/multipotent stem cells would help in improve developmental models, improve directed differentiation into adult cells and allow more efficient and less costly approaches to conventional methods.
Human pluripotent cells (such as human embryonic stem cells [hESCs] and induced pluripotent stem cells [iPS cells]) can be differentiated through a bi-potential mesendoderm (T+, MixL1+) precursor that can be further differentiated into a wide range of mesoderm lineages such as bone, blood, muscle and kidney. See FIG. 12. Different types of mesoderm precursors can be formed in embryonic development from mesendoderm. These include lateral plate mesoderm, splanchnic mesoderm, paraxial mesoderm and somatic mesoderm. Each of these mesoderm precursors gives rise to different types of mesoderm tissue (FIG. 12). IMP cells represent Isl1+ Nkx2.5+ splanchnic mesoderm, a type of mesoderm that forms the cardiovascular system and hematopoietic system.
The epicardium is derived from Isl1+ splanchnic mesoderm and constitutes the outer layer of the vertebrate heart. Embryologicaly, the epicardium is derived from a source of pro-epicardium though to originate in the septum traversum. Epicardium consists of a single layer of flat mesothelium that is connected to the mycocardium by sub-epicardial connective tissue (Manner et al., 2001, Cells Tissues Organs 169, 89-103). Formation of the epicardium over the developing heart coincides with the development of coronary blood vessels (Olivey et al., Trends in Cardiovasc Med 2004, 14, 247-251). Once the pro-epicardium comes into contact with the developing heart at around the time of beating, it spreads over the myocardium forming a new layer, the epicardium. The epicardium and related cells/tissue preceding the epicardium then gives rise to multiple cell types that together make up the coronary vasculature including smooth muscle cells, endothelial cells and cardiac fibroblasts. See FIG. 36. Epicardial cells also have the capacity to differentiate into cardiomyocytes (Zhou et al., 2008 Nature 454, 109-113). Soon after invading the myocardial surface, sub-populations of epicardial cells undergo an epithelial to mesenchymal transition and migrate into the sub-epicardial space. Some of these cells then have the capacity to further migrate into the compact zone of the myocardium. Coronary blood vessels form as angioblasts, derived from epicardium and/or other cells migrating into the heart, coalesce to form a primitive vascular plexus in the sub-epicardial space and in the myocardium. Eventually, these endothelial tubes coalesce to form larger vessels that become the coronary arteries and veins. The complement of cells comprising the coronary vasculature including smooth muscle and endothelial cells and, interspersed fibroblasts—all originating from progenitors in the pro-epicardium/epicardium. Epicardium is typically signified by expression of Wilm's tumor rotein 1 (WT1), T-box factor 18 (Tbx18), epicardin (Tcf21) and RALDH2 (Zhou et al., 2008; Cai et al., 2008, Nature 454, 104-108). The WT1+ epicardium is believed to form from an Isl1+ Nkx2.5+ precursor (Zhou et al., 2008). Progenitor cells expressing Wt1+ originating from the pro-epicardium/epicardium contribute to formation of the coronary vasculature. The EPC described herein represents a coronary vascular progenitor cell derived from human pluripotent cells.
As another aspect of this invention, conditions for the differentiation of human pluripotent cells into multipotent migratory cells (MMCs) have been described. MMCs form directly from adherant pluripotent cells in chemically defined media. MMCs are generated by treating human pluripotent cells with small molecule compounds to culture media. See FIG. 13. These compounds are known inhibitors of GSK3 activity (BIO) and TGFβ/Activin A/Nodal signaling (SB431542). By further treatment, MMCs can be differentiated into a wide range of cell types. By other treatments, MMCs can be converted to a CXCR4+ CD56+ population of cells (C56Cs, for CXCR4+/CD56+ cells), that up-regulate additional cell surface markers. In addition to expressing the cytokine receptor CXCR4 and CD56, C56Cs can up-regulate the stem cell marker c-Kit. C56Cs do not express markers for hematopoietic stem cells, such as CD45, or endothelial markers such as CD31.
Since C56Cs are produced from MMCs and express markers for receptors of cytokine signaling (CXCR4) known to be involved in stem cell ‘homing’ to ischemic-inflammatory tissue, it is possible that these cells may be capable of ‘homing’ to sites of tissue damage. Systemic administration by intravenous administration would be one way whereby these cells could home to damaged tissue and participate in repair processes. Once these cells have homed to damaged tissue, they may then promote tissue repair by paracrine mechanisms or by trans-differentiating into cells that participate directly in repair. These cells may also participate in the suppression of inflammatory responses and by immuno-modulation (suppressing T cells, natural killer cell activity).